FUMADORES

The integrated resonances for selected metabolite peaks were plotted over time to investigate food-related metabolite excretion kinetics. This was done in order to illustrate the delay of metabolite excretion following food ingestion.

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3.5.3.1 Metabolite excretion following fruit consumption

Figure 10 shows the excretion kinetics of the metabolites hippurate, tartrate, proline betaine and 4-OH-hippurate. Proline betaine and tartrate excretion occurred directly after fruit consumption (before bedtime of day 2) and tartrate re-occurred after wine and grapes consumption. The excretion of 4-OH-hippurate after the fruit meals was delayed in comparison and started in the morning of day 3. Hippurate showed high inter-individual variation at each time point and the excretion was relatively higher after fruit consumption. In addition, there were apparent diurnal variations, as seen by low hippurate excretion in the lunchtime sample. However, the timing of the standard food consumption, with whole-grain bread as fibre sources being served for breakfast and lunch, might have biased this observation.

Figure 10 Metabolite plot showing the mean peak integral and its standard deviation of hippurate, tartrate, proline betaine and 4-OH-hippurate over time. The time of food consumption is indicated in red. Tartrate and proline betaine are both immediately excreted after fruit excretion while 4-OH-hippurate appears 12 hours post fruit consumption. The second tartrate peak (end of day 4) occurred after consumption of wine and grapes. Excretion of hippurate showed apparent high diurnal variation and high standard deviation.

3.5.3.2 Metabolite excretion following animal protein consumption

Consumption of animal protein (fish and/or beef) lead to increased excretion of the metabolites TMAO, creatine, taurine, an unknown metabolite at δ 2.90 (t), choline and carnitine (Figure 11). The unknown metabolite with chemical shift at δ 2.90 (t) showed further resonances at δ 2.13 (m), δ 2.52 (m) and δ 4.43 (dd), as ascertained by COSY and

TOCSY, and corresponding 13C carbon shifts are listed in the appendix. Besides choline and carnitine, there were other resonances increased in the spectral area δ 3.21 – 3.33, all subject to peak shifts. These resonances might derive from ethanolamine, other amines and choline glycerophospholipids [151]. The integrals of the spectral areas δ 3.21 – 3.26 and δ 3.29 – 3.33 were therefore chosen to illustrate these metabolites. After fish intake, all of the plotted metabolites were excreted in the next collected urine sample. Beef intake did not result in immediately increased excretion of TMAO and metabolites in the spectral area δ 3.29 – 3.33, but the metabolites creatine, taurine, δ 2.90 (t) and resonances in the spectral area δ 3.21 – 3.26 were excreted immediately afterwards. Another in-house nutritional study, where only beef consumption was investigated showed that TMAO excretion occurs after beef consumption but is delayed by up to 12 hours. This suggests the possibility that endogenous or gut microbial metabolism is necessary to produce TMAO from beef sources. Interestingly, taurine excretion is slightly elevated in every night sample. This is likely to be due to consumption of a ham sandwich every day as part of the lunch meal, considering that ham is a good source of taurine. Apparent excretion of metabolites in the spectral area δ 3.29 – 3.33 at the end of day 2 and beginning of day 3 derives from peak overlap with proline betaine (proline betaine CH3 moiety δ 3.31).

Figure 11 Metabolite plot showing the mean peak integral and its standard deviation of (A) TMAO, creatine and spectral areas of δ 3.21 – 3.26 and δ 3.29 – 3.33 and (B) of δ 2.90 (t) and taurine. Creatine, δ 2.90 (t) and taurine are observed in the urine collection within 2 hours post fish and beef consumption. TMAO excretion starts immediately after fish consumption but is delayed after beef consumption and therefore overlaps with the second fish meal. Metabolites in the spectral area of δ 3.21 – 3.26 get excreted after fish and beef consumption and metabolites in the spectral area δ 3.29 – 3.33 are excreted immediately only after fish excretion and might be delayed after beef consumption. The proline betaine peak (δ 3.31) is overlapped in the spectral region δ 3.29 – 3.33 (see end of day 2). The time of food consumption is indicated by the red arrows.

3.5.3.3 Changes in metabolite excretion following consumption of wine and grapes

Excretion kinetics of metabolites following wine and grape consumption on day 4 were investigated (Figure 12). Ethanol, δ 1.10 (d) and 2-isophenylmalate were present only in

the urine sample immediately following wine consumption. The amount of ethanol and δ 1.10 (d) excretions were highly variable between the individuals. Tartrate, 2,3- butanediol and diethylmalonate were excreted up to 12 hours following wine and grape consumption.

Figure 12 Metabolite plot showing the mean peak integral and its standard deviation of (A) ethanol, 2- isophenylmalate and δ 1.10 (d) and (B) tartrate, 2,3-butanediol and diethylmalate. All 6 metabolites are immediately excreted after wine + grape consumption. The tartrate peak at the end of day 2 derives from consumption of grapes present in the fruit mix.

3.5.4 1H NMR spectra of the tested foods, food extracts and beverages

To assign the origin of several elucidated urinary metabolites after the food challenges, all foods (fish, beef, orange, apple, grapes, grapefruit and wine) were analysed using

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H-HR-MAS or 1H NMR spectroscopy. The origin of proline betaine in urine spectra could be assigned to proline betaine in orange and grapefruit, excreted tartrate was present due to ingestion of grapes and wine in the diet. Furthermore, wine and fruits were sources of polyphenols. Ethanol, 2,3-butanediol, 2-isophenylmalate, diethylmalonate and an unknown compound with chemical shifts at δ 1.10 (d) and 3.90 (m) were present in wine. Taurine, choline, carnitine and creatine were present in fish and beef, TMAO and anserine were only present in fish and carnosine was only present in beef. It is well established, that anserine and carnosine are found in fish and beef [152]. The fatty acid pattern differed between fish and beef, with the fish spectrum showing an additional resonance at δ 2.80 ppm. Spectra of these foods are shown in Figure 13-15.

Figure 14 1H NMR spectra of juices from (A) apple, (B) grapefruit and (C) orange. The aromatic region (δ 9.5 – 5.45 ppm) is vertically enlarged by x 280 and the aliphatic region (δ 3.21 – 0.5 ppm) is vertically enlarged by x10. Abbreviations: BCAA, branch-chain amino acids; GABA, 4-aminobutyrate, Asn, asparagine; Asp, aspartate.

Figure 15 1H NMR spectrum of (A) grape juice and (B) red wine. The grape juice spectrum is vertically enlarged in the aromatic region by x400 and by x25 on the aliphatic region. The aromatic region of red wine spectrum is vertically enlarged by x77. Abbreviations: BCAA, branch-chain amino acids; GABA, 4- aminobutyrate.

3.5.5 Confirmatory study to proline betaine excretion after orange juice